Illumination for fluorescence imaging using objective lens

ABSTRACT

A system includes: an objective lens; a first light source to feed first illuminating light through the objective lens and into a flowcell (e.g., with a relatively thin film waveguide) to be installed in the system, the first illuminating light to be fed using a first grating on the flowcell; and a first image sensor to capture imaging light using the objective lens, wherein the first grating is positioned outside a field of view of the first image sensor. Dual-surface imaging can be performed. Flowcells with multiple swaths bounded by gratings can be used. An auto-alignment process can be performed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a non-provisional of, and claims the benefit of thefiling date of, U.S. provisional application 62/649,996, filed Mar. 29,2018, the contents of which are incorporated herein by reference.

BACKGROUND

High-throughput DNA sequencing can be the basis for genomic analysis andother genetic research. In this and other types of sequencing,characteristics of a sample of genetic material are determined byilluminating the sample, for example using a planar waveguide. Sometypes of image detection can place particular demands on the sequencingsystem in order to create an image of the entire sample. For example,with a time-delay and integration (TDI) sensor the image is captured onenarrow sliver at a time, involving a scanning operation along the lengthof the slide. To reduce photodamage to the sample from the illuminatinglight, it is often desirable to confine the illumination to the samplearea being imaged. In some instances, this creates the challenge ofensuring that the illumination is synchronized with the image detectionthroughout the scanning process.

SUMMARY

In a first aspect, a system includes: an objective lens; a first lightsource to feed first illuminating light through the objective lens andinto a flowcell to be installed in the system, the first illuminatinglight to be fed using a first grating on the flowcell; and a first imagesensor to capture imaging light using the objective lens, wherein thefirst grating is positioned outside a field of view of the first imagesensor.

Implementations can include any or all of the following features. Theflowcell is installed in the system. The first grating and a secondgrating are positioned on the flowcell. The first and second gratingshave different coupling angles. The first and second gratings havedifferent grating periods. The flowcell includes a first sample surfaceparallel to a second sample surface, the first grating to couple a firstportion of the first illuminating light to illuminate the first samplesurface, and the second grating to couple a second portion of the firstilluminating light to illuminate the second sample surface. The systemfurther comprises a first planar waveguide into which the first gratingcouples the first portion of the first illuminating light, and a secondplanar waveguide into which the second grating couples the secondportion of the first illuminating light. The first grating is offset,relative to the second grating, in a travel direction of the firstportion of the first illuminating light. The first and second gratingsare positioned on opposite sides of a planar waveguide that illuminatesthe flowcell, the first grating coupling the first illuminating lightinto the planar waveguide, and the second grating coupling the firstilluminating light out of the planar waveguide. The system furthercomprises a wall that blocks the first illuminating light coupled out ofthe planar waveguide by the second grating from entering the objectivelens. The first illuminating light comprises a first light beam of afirst wavelength, the system further comprising a second light source tofeed second illuminating light through the objective lens, the secondilluminating light comprising a second light beam of a secondwavelength. The second light source is to feed the second illuminatinglight through the objective lens into the flowcell via the firstgrating, the first grating having a symmetric coupling angle for thefirst and second wavelengths. The first light source is directing thefirst light beam at a first side of the objective lens, and the secondlight source directing the second light beam at a second side of theobjective lens opposite the first side. The system further comprises afirst mirror and a tube lens positioned after the first and second lightsources and before the objective lens, wherein respective angles of thefirst and second light beams in propagation from the first mirror to thetube lens reflect corresponding incident angles of the first and secondlight beams on the first grating. The system further comprises a secondmirror positioned after the first light source and before the firstmirror, the second mirror to provide a spatial separation of the firstand second light beams on the first grating. The flowcell has multipleswaths bounded by respective gratings including the first grating, andwherein the system uses at least one of the respective gratings both asan entry grating and an exit grating. A waveguide material of theflowcell includes Ta₂O₅. The first image sensor comprises a time delayand integration sensor, the system further comprising a moveable stageholding the flowcell. The system further comprises a thermal stage tohold the flowcell, the thermal stage providing thermal control. Thesystem further comprises a second image sensor that captures images ofat least the first grating and a planar waveguide in the flowcell,wherein the system evaluates the images using an alignment criterion.The light has a wavelength to trigger a fluorescent response from asample in the flowcell.

In a second aspect, a flowcell includes: a substrate to hold a sample; afirst planar waveguide to lead first light for the sample; a firstgrating to couple the first light; a second planar waveguide to leadsecond light for the sample; and a second grating to couple the secondlight.

Implementations can include any or all of the following features. Thefirst grating is positioned on the first planar waveguide, and thesecond grating is positioned on the second planar waveguide. The firstand second gratings have different coupling angles. The first and secondgratings have different grating periods. The flowcell includes a firstsample surface parallel to a second sample surface, the first grating tocouple the first light to illuminate the first sample surface, and thesecond grating to couple the second light to illuminate the secondsample surface. The first grating is offset, relative to the secondgrating, in a travel direction of the first light. The first grating tocouple the first light into the first waveguide, the second grating tocouple the second light into the second waveguide, the flowcell furthercomprising a third grating to couple the first light out of the firstplanar waveguide, and a fourth grating to couple the second light out ofthe second planar waveguide.

In a third aspect, a method includes: feeding illuminating light throughan objective lens and into a flowcell using a first grating positionedoutside an image-sensor field of view; and capturing imaging light usingthe objective lens.

Implementations can include any or all of the following features. Theflowcell includes a first sample surface parallel to a second samplesurface, the method further comprising directing a first component ofthe illuminating light to a first grating aligned with the first samplesurface, and directing a second component of the illuminating light to asecond grating aligned with the second sample surface. The methodfurther comprises adjusting the objective lens to focus on the firstsample surface in connection with directing the first component of theilluminating light to the first grating, and adjusting the objectivelens to focus on the second sample surface in connection with directingthe second component of the illuminating light to the second grating.The illuminating light comprises a first light beam of a firstwavelength, and a second light beam of a second wavelength, the methodfurther comprising directing the first light beam at a first side of theobjective lens, and directing the second light beam at a second side ofthe objective lens opposite the first side. The method further comprisesblocking the illuminating light, upon the illuminating light exiting theflowcell, from entering the objective lens. A thermal stage holds theflowcell, the method further comprising providing thermal control of theflowcell using the flowcell. The flowcell has multiple swaths bounded byrespective gratings including the first grating, the method furthercomprising using at least one of the respective gratings both as anentry grating and an exit grating. The method further comprisesperforming an alignment process that evaluates a quality of coupling bythe first grating.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein. In particular, allcombinations of claimed subject matter appearing at the end of thisdisclosure are contemplated as being part of the inventive subjectmatter disclosed herein.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an example of an optical layout for planar waveguideillumination.

FIG. 2 shows an example of an objective lens.

FIG. 3 shows an example of a flowcell with a waveguide and a grating.

FIG. 4 shows an example of coupling of a laser beam into and out of aplanar waveguide using gratings.

FIG. 5 shows an example graph of coupling angle versus grating period.

FIG. 6 shows an example graph of coupling angle tolerance versus beamdiameter for multiple wavelengths.

FIG. 7 shows an example graph of simulated angle tolerance versus beamwaist.

FIG. 8 shows an example graph of intensity across a waveguide.

FIG. 9 shows an example image constructed from line scans.

FIG. 10 shows an example image of fluorescence dye molecules.

FIG. 11 shows an example image of a laser beam shape within a waveguide.

FIGS. 12-15 show example images of clusters of genetic material.

FIG. 16 schematically shows an example of a system for laserillumination and fluorescence imaging.

FIG. 17 shows an example of dual surface imaging using a flowcell.

FIGS. 18-19 show other examples of dual surface imaging using aflowcell.

FIG. 20 shows an example image of flowcell illumination.

FIGS. 21A-21B show example flowcharts of a process.

FIG. 22 schematically shows an example of a system for laserillumination and fluorescence imaging.

FIG. 23 shows an example flowchart of a process for alignment.

FIG. 24 shows an example of a flowcell with multiple swaths.

DETAILED DESCRIPTION

This document describes examples of illumination for fluorescent imagingusing an objective lens. In some implementations, an opticalarchitecture can facilitate low-power, low-background, illumination forhigh-speed scan imaging. This can involve synchronizing the illuminationwith the detection through the microscope objective lens. At asignal-to-noise ratio (SNR) equivalent to that of traditional planarwaveguide illumination, a laser power reduction of up to about 70 timescan be obtained using such an approach.

In some implementations, laser beams can travel through the objectivelens so as to synchronize the imaging optics and the illumination witheach other. The waveguide and one or more gratings can be designed withcoupling angles needed by the numerical aperture of the objective lens,and by the wavelength of the laser. Laser beam shaping optics can beused to define proper beam dimensions and the quality of light to assurecoupling efficiency and tolerance. A proper waveguide, grating, claddingand/or substrate material can be selected to be compatible with thesequencing chemistry and the runs being performed. A proper geometry andoptical performance for fluorophore excitation with low background canbe designed. In some implementations, dual-surface imaging can besupported. In some implementations, multiple-color excitation (e.g.,dual-color) can be supported.

Delivering illuminating light such as one or more laser beams throughthe imaging objective lens can provide advantages over existingapproaches. For example, providing the laser beam through the objectivelens means that illumination and image capture are performed on the sameside of the flowcell. The backside of the flowcell then can be used forfacilitating one or more other aspects of the process, such as accurateor rapid temperature control of the sample. Fine control of theillumination laser beam with regard to size, angle and position can beprovided. This can increase the alignment tolerance on the grating,which in turn can significantly reduce the cost of optomechanics andgrating fabrication. High-speed scanning can be facilitated byco-registration of an illumination line with the detection apparatus(e.g., a TDI sensor). Optics can be designed to shape the laser beam asa narrow line formed inside a planar waveguide. For example, the planarwaveguide can have a thickness range of about 50-200 nanometers (nm) orlarger. As such, the planar waveguide can be considered a relativelythin film waveguide. A planar waveguide can provide a high power densitywith a relatively low amount of laser power. Materials compatible withthe surface and the sequencing chemistry (including, but not limited to,Ta₂O₅ or SiN) can be used. In some implementations, Ta₂O₅ can be used asa core, SiO₂ can be used as a substrate, and a water buffer or a polymerlayer can be used as cladding. For example, the cladding can have arefractive index of about 1.3-1.5. Grating couplers can be designed tohave different acceptance angles for different wavelengths. For example,red and green laser lights can be used. As another example, gratingcouplers can be designed on different surfaces (e.g., top and bottomsurfaces) of the flowcell.

Examples described herein relate to sequencing of genetic material.Sequencing can be performed on a sample to determine which buildingblocks, called nucleotides, make up the particular genetic material thatis in the sample. The sequencing can be done after the genetic materialhas first been purified and then replicated a number of times so as toprepare a sample of a suitable size.

Imaging can be performed as part of the process of sequencing thegenetic material. This can involve fluorescent imaging, where a sampleof genetic material is subjected to light (e.g., a laser beam) totrigger a fluorescent response by one or more markers on the geneticmaterial. Some nucleotides of the genetic material can have fluorescenttags applied to them, which allows for determination of the presence ofthe nucleotide by shining light onto, and looking for a characteristicresponse from, the sample. Fluorescent responses can be detected overthe course of the sequencing process and used to build a record ofnucleotides in the sample.

Examples described herein refer to flowcells. A flowcell is a substratethat can be used in preparing and carrying one or more samples in atleast one stage of a sequencing process. The flowcell is made of amaterial that is compatible with both the illumination and the chemicalreactions to which it will be exposed. The substrate can have one ormore channels in which sample material can be deposited. A substance(e.g., a liquid) can be flowed through the channel where the samplegenetic material is present to trigger one or more chemical reactionsand/or to remove unwanted material. The flowcell may enable the imagingby facilitating that the sample in the flowcell channel can be subjectedto illuminating light and that any fluorescent responses from the samplecan be detected. Some implementations of the system may be designed tobe used with at least one flowcell, but may not include the flowcell(s)during one or more stages, such as during shipping or when delivered toa customer. For example, the flowcell(s) can be installed into animplementation at the customer's premises in order to perform analysis.

Examples herein refer to coupling of light (e.g., a laser beam) intoand/or out of a waveguide by one or more gratings. A grating can couplelight impinging on the grating by way of diffracting at least a portionof the light, thereby causing the portion of the light to propagate inone or more other directions. In some implementations, the coupling caninvolve one or more interactions, including, but not limited to,reflection, refraction and/or transmission of the portion of the light.

FIG. 1 shows an example of an optical layout 100 for planar waveguideillumination. The optical layout 100 can be implemented as part of oneor more systems described herein. The optical layout 100 can be used forperforming one or more techniques or processes described herein.

The optical layout 100 can include one or more light sources. Forexample, light sources can provide light having one or more wavelengths.In one implementation described herein, the optical layout 100 includesa laser 102 and a laser 104. In some implementations, the lasers 102 and104 can be characterized as a green laser and a red laser, respectively.For example, the laser 102 can generate light that has one or morewavelengths in the range of about 400-570 nm. For example, the laser 104can generate light that has one or more wavelengths in the range ofabout 620-750 nm. One or more other types of light (e.g., a differentwavelength) can instead or additionally be used in some implementations.

Light from either or both of the lasers 102 and 104 can be directed atleast one mirror in the optical layout 100. A mirror can be made fromone or more materials that provide a partial or total reflection of thetype(s) of light generated by the relevant light source. For example, atransparent substrate can be provided with a mirror coating. Here, amirror 106 is used for the light beam from the laser 102. Prism mirrorscan be used to combine two beams with a small angle difference. Dichroicmirrors can be used to combine two/three different wavelengths. Mirrorscan be mounted on actuated stages for automatic alignment.

The light beam(s) generated in the optical layout 100 can be shaped orotherwise conditioned in one or more ways. In some implementations, oneor more beam-shaping optics 108 can be used. For example, thebeam-shaping optics 108 can serve to provide an aperture for the lightbeam, to transform the light beam, and/or to integrate components of thelight beam in one or more ways. A separate one of the beam-shapingoptics 108 (identical ones or different ones) can be provided for thelight beams of the lasers 102 and 104, respectively.

The light beam(s) shaped by the beam-shaping optics 108 can be directedat least one mirror in the optical layout 100. Here, a mirror 110 isused for the light beams of both the lasers 102 and 104. The mirror 106can be adjusted to control the direction of the light beam(s). In someimplementations, a coupling angle of the light beam at an object planecan be adjusted using the mirror 106. For example, the object plane canbe defined at a component (e.g., a grating) that is positioneddownstream of the light beam(s) in the optical layout 100. The mirror110 can be adjusted using one or more actuators. In someimplementations, the mirror 110 is electrically adjustable. For example,the mirror 110 can be actuated using one or more piezoelectric motors.

The optical layout 100 can include one or more components for focusingthe light from at least one light source. In some implementations, atube lens 112 can be used. For example, the tube lens can be used tofocus the light beam(s) into an intermediate image that is provided toan infinity corrected objective in the optical layout 100. Here, thetube lens 112 receives the light beams that originated at the lasers 102and 104, respectively.

The mirror 110 can be placed in a position selected based on one or moreother components in the optical layout 100. In some implementations, theplacement of the mirror 110 can depend at least in part on thecharacteristics and location of the tube lens 112. For example, themirror 110 can be placed at a back focal plane 114 of the tube lens 112with regard to an object plane, such as a planar waveguide at aflowcell.

That is, light generated by the laser 102 can form a laser beam 116 thatpropagates from the mirror 110 toward the tube lens 112. Similarly,light generated by the laser 104 can form a laser beam 118 thatpropagates from the mirror 110 toward the tube lens 112. Each of thelaser beams 116 and 118 can be generated so as to propagate in the sameor a different direction. In some implementations, the direction(s) canbe characterized with regard to an optical axis 120 of the tube lens112. For example, the laser beam 116 can form an angle 122 with regardto the optical axis 120. For example, the laser beam 116 can form anangle 124 with regard to the optical axis 120. The angles 122 and 124can be selected so as to provide a specific coupling angle (the same ordifferent) for the respective laser beams 116 and 118 at a downstreamcomponent, such as a grating.

The tube lens 112 can direct the laser beams 116 and 118 at one or morecomponents in the optical layout 100. In some implementations, the tubelens 112 directs the light at a mirror 126 that reflects at least partof the light toward an objective lens 128 in the optical layout 100. Forexample, the mirror 126 can be a dichroic mirror having thecharacteristic that it reflects into the objective lens 128 asignificant portion of incident light having the wavelength(s) of thelasers 102 and 104, while also having the characteristic of transmittinga significant portion of light having one or more other wavelengths. Thereflected portion of the light from the lasers 102 and 104 can be atleast an amount such that the reflected light sufficiently illuminates adownstream component, such as a planar waveguide of a flowcell. In otherimplementations, the optical layout 100 can instead be arranged for themirror 126 to at least in part transmit the illuminating light (i.e.,the light originating at the lasers 102 and 104) into the objective lens128, and at least partially reflect other light emerging from theobjective lens 128.

The objective lens 128 can include one or more lenses and/or otheroptical components. In some implementations, the objective lens 128 caninclude the components shown in FIG. 2. The objective lens 128 can serveto direct illuminating light at one or more components of the opticallayout 100. The objective lens 128 can serve to capture imaging lightand provide it to one or more components of the optical layout 100.

The optical layout 100 includes a flowcell 130. In some implementations,the flowcell can include any flowcell described or shown elsewhereherein. For example, the flowcell 130 can include one or more channels132 configured to hold sample material and to facilitate actions to betaken with regard to the sample material, including, but not limited to,triggering chemical reactions or adding or removing material. Here, across section of the channel 132 in a longitudinal direction is shown.That is, if a liquid is flowed through the channel 132, the main flowdirection of the liquid can be into or out of the drawing in thisillustration.

An object plane 134 can be defined with regard to another component inthe optical layout 100. Here, the object plane 134 is defined at leastin part by the objective lens 128. In some implementations, the objectplane 134 extends through the flowcell 130. For example, the objectplane 134 can be defined so as to be adjacent the channel(s) 132.

The objective lens 128 can define a field of view 136. The field of view136 can define the area on the flowcell 130 from which an image detectorcaptures imaging light using the objective lens 128. One or more imagedetectors can be used. For example, when the lasers 102 and 104 generaterespective laser beams having different wavelengths (or differentwavelength ranges), the optical layout 100 can include separate imagedetectors for the respective wavelengths (or wavelength ranges).

One or more diffraction gratings can be placed in relation to theobjective lens 128. In some implementations, gratings 138 and 140 can beplaced at or near the object plane 134. For example, the gratings 138and 140 can be placed on opposite sides of the channel 132. That is, thegratings 138 and 140 can be placed so as to define a transversedirection across the channel 132, perpendicular to a longitudinaldirection of the channel 132. The gratings 138 and 140 are placedoutside the sensor field of view 136.

The gratings 138 and 140 can direct light into and/or out of a planarwaveguide in the flowcell 130. Here, beams 142 and 144 are incident onthe grating 138. In some implementations, the beams 142 and 144 cancorrespond to the laser beams 116 and 118, respectively, and can definean illumination area in the flowcell 130 (e.g., in a planar waveguidewithin the flowcell 130). For example, the beam 142 and the beam 144 canbe incident on the grating 138 at different coupling angles. As anotherexample, the beam 142 and the beam 144 can be incident on the grating138 at different locations in the longitudinal direction of the channel132. Here, beams 146 and 148 are emerging from the grating 140. Forexample, the beam 146 can correspond to at least a portion of the beam142 after having traversed a planar waveguide in the flowcell 130. Forexample, the beam 148 can correspond to at least a portion of the beam144 after having traversed a planar waveguide in the flowcell 130.

The gratings 138 and 140 can be identical or similar to each other, orcan be different types of gratings. The grating(s) can include one ormore forms of periodic structure. In some implementations, the grating138 and/or 140 can be formed by removing or omitting material from theflowcell 130 (e.g., from a waveguide material that is included in theflowcell 130). For example, the flowcell 130 can be provided with a setof slits and/or grooves therein to form the grating 138 and/or 140. Insome implementations, the grating 138 and/or 140 can be formed by addingmaterial to the flowcell 130 (e.g., to a waveguide material that isincluded in the flowcell 130). For example, the flowcell 130 can beprovided with a set of ridges, bands or other protruding longitudinalstructures to form the grating 138 and/or 140. Combinations of theseapproaches can be used.

Within the field of view 136, the objective lens 128 can capture imaginglight from the flowcell 130. For example, the imaging light can includeluminescence. This imaging light propagates through the objective lens128 and emerges at the other end thereof. The optical layout 100 can useone or more components to direct the imaging light in a differentdirection that that from which the illuminating light (i.e., the lightfrom the lasers 102 and 104) arrived. Here, the mirror 126 can be adichroic mirror. For example, a dichroic mirror can reflect at leastpart of the imaging light and transmit at least part of the illuminatinglight. Instead, a dichroic mirror can transmit at least part of theimaging light and reflect at least part of the illuminating light. Asignal 150 here represents transmission of the imaging light to one ormore image sensors 152. In some implementations, the imaging lightincludes fluorescence light from the flowcell 130. For example, theimage sensor 152 can include a time delay and integration (TDI) sensor.

The objective lens 128 can be chosen to have a numerical aperture thatallows the grating 138 and/or 140 to be illuminated outside of the fieldof view 136. In some implementations, the numerical aperture is lessthan one. For example, numerical apertures of 0.95, 0.75, 0.7, 0.3and/or 0.2 can be used. In some implementations, a greater numericalaperture can be used with water/oil immersion objective lenses, forexample a numerical aperture of 1.2 to 1.4 or greater.

In some implementations, the size of the flowcell 130 can necessitatecapturing of multiple images by scanning along the longitudinaldirection (into and/or out of the drawing) of the channel 132. Theillumination light (e.g., the beams 142 and 144) can then beco-registered with the field of view 136 by virtue of the objective lens128 both directing the illuminating light from the lasers 102 and 104 atthe grating 138 and/or 140, and defining the field of view 136. That is,the lasers 102 and/or 104, the objective lens 128 and the grating 138and/or 140 can serve for co-registering an illumination area on theflowcell 130 with the field of view 136 of the image sensor on theflowcell 130, at successive locations on the flowcell.

The optical layout 100 is an example of a system that includes anobjective lens (e.g., the objective lens 128); a light source (e.g., thelasers 102 and 104) to feed illuminating light (e.g., the laser beams116 and 118) through the objective lens and into a flowcell (e.g., theflowcell 130) to be installed in the system, the illuminating light tobe fed using a grating (e.g., the grating 138) on the flowcell; and animage sensor to capture imaging light using the objective lens, whereinthe grating is positioned outside a field of view (e.g., the field ofview 136) of the image sensor.

The optical layout 100 is an example of a system that can be used withfirst and second gratings (e.g., the gratings 138 and 140) positioned ona flowcell (e.g., the flowcell 130).

The optical layout 100 is an example of a system where firstilluminating light from a first light source (e.g., the laser 102)comprises a first light beam (e.g., the laser beam 116) of a firstwavelength, and where the system further comprises a second light source(e.g., the laser 104) to feed second illuminating light through anobjective lens, the second illuminating light comprising a second lightbeam (e.g., the laser beam 118) of a second wavelength.

The optical layout 100 is an example of a system that includes a mirror(e.g., the mirror 110) and a tube lens (e.g., the tube lens 112)positioned after first and second light sources (e.g., the lasers 1102and 104) and before an objective lens (e.g., the objective lens 128),wherein respective angles of the first and second light beams (e.g., theangles 122 and 124) in propagation from the mirror to the tube lensreflect corresponding incident angles of the first and second lightbeams on the grating (e.g., the respective coupling angles of the beams142 and 144).

The optical layout 100 is an example of a system having a mirror (e.g.,the mirror 106) positioned after a light source (e.g., the laser 102)and before a mirror (e.g., the mirror 110), the mirror to provide aspatial separation of the first and second light beams on the grating(e.g., the different incidence locations on the grating 138 of the beam142 and the beam 144 in the longitudinal direction of the channel 132).

FIG. 2 shows an example of an objective lens 200. The objective lens 200can include multiple lenses 202 arranged (here in a coaxial manner) soas to allow transmission of light in both directions and to manipulatethe light in one or more ways. The objective lens 200 is here directedat a flowcell 204. For example, the flowcell 204 can have one or moregratings that allow coupling of light into and/or out of a planarwaveguide in the flowcell 204.

The objective lens 200 can be used with one or more light sources. Here,a laser 206 is positioned at one end of the objective lens 200. In someimplementations, the laser 206 generates a laser beam 208 into theobjective lens 200. For example, the laser beam 208 can have awavelength of about 532 nm. Here, a laser 210 is positioned at the sameend of the objective lens 200. In some implementations, the laser 210generates a laser beam 212 into the objective lens 200. For example, thelaser beam 212 can have a wavelength of about 660 nm. The laser beam 208can be directed so as to propagate at or near a side 214 of theobjective lens 200. The laser beam 212 can be directed so as topropagate at or near a side 216 of the objective lens 200 that isopposite the side 214.

In some implementations, the laser beams 208 and 212 are incident onrespective gratings on the flowcell 204. In some implementations, thegrating where the laser beam 208 enters the planar waveguide can be thegrating where the laser beam 212 exits the planar waveguide, the laserbeam 212 having entered the planar waveguide at its correspondinggrating. Correspondingly, the grating where the laser beam 212 entersthe planar waveguide can be the grating where the laser beam 208 exitsthe planar waveguide, the laser beam 208 having entered the planarwaveguide at its corresponding grating. If no blocking is done, theexiting light beams can propagate into the objective lens 200.

The above example illustrates that a system can include a first lightsource (e.g., the laser 206) directing a first light beam (e.g., thelaser beam 208) at a first side (e.g., the side 214) of an objectivelens (e.g., the objective lens 200), and a second light source (e.g.,the laser 210) directing a second light beam (e.g., the laser beam 212)at a second side (e.g., the side 216) of the objective lens opposite thefirst side. The above example illustrates that a system can provideilluminating light that comprises a first light beam of a firstwavelength (e.g., the laser beam 208), and a second light beam of asecond wavelength (e.g., the laser beam 212), and that the first lightbeam can be directed at a first side (e.g., the side 214) of anobjective lens (e.g., the objective lens 200), and that the second lightbeam can be directed at a second side (e.g., the side 216) of theobjective lens opposite the first side.

The above example illustrates that a system (e.g., including theobjective lens 200 and the lasers 206 and 210) can be used with firstand second gratings positioned on opposite sides of a planar waveguidethat illuminates a flowcell (e.g., the flowcell 204), the first gratingcoupling first illuminating light (e.g., the laser beam 208 or 212) intothe planar waveguide, and the second grating coupling the firstilluminating light out of the planar waveguide.

FIG. 3 shows an example of a portion 300 of a flowcell. The flowcell hasa waveguide 302 and a grating 304, each of which is partially shown inthe portion 300 of the flowcell. The material(s) for the waveguide 302can include Ta₂O₅, having a refractive index of about 2.1. For example,the waveguide 302 can have a thickness of about 100-200 nm. A specifiedthickness for the waveguide 302 can have a tolerance of about ±2.5 nm insome implementations. An area 306 of the flowcell 300 (e.g., a claddingpositioned above the waveguide 302 and the grating 304 in this view) canhave a refractive index comparable to that of water. An area 308 of theflowcell 300 (e.g., a substrate positioned below the waveguide 302 andthe grating 304 in this view) can have a refractive index comparable tothat of certain glass. In some implementations, the area 308 can includethe material glass substrate with a refractive index of about 1.5. Forexample, the area 308 can have a thickness of about 300 micrometers(μm). The grating 304 can include SiO₂ having a grating thickness ofabout 10-100 nm. A specified grating thickness for the grating 304 canhave a tolerance of about ±2.5 nm. For example, the grating 304 can havea duty cycle of about 50±10%.

The illustration shows a design model for the flowcell 300. In someimplementations, the image showing the flowcell 300 can be generatedusing finite-difference time-domain analysis. For example, the image canbe generated using a software system that provides physics modeling.

In the flowcell 300, incident light (e.g., a laser beam) enters asindicated by an arrow 310 and is at least in part refracted by thegrating 304. The refracted portion of the light propagates in thewaveguide 302. The coupling angle depends on the (laser) wavelength,grating pitch/depth, and the refractive index. Intensity of light isindicated by shading according to a scale 312. A maximum couplingefficiency can be about 40%. The design can be chosen so that symmetriccoupling angles or preferred angles by optical platform are defined forlight of multiple wavelengths. For example, the grating 304 can have oneor more symmetric coupling angles for a red laser beam and a green laserbeam.

In some implementations, a grating can be made from SiO₂. In someimplementations, a grating can be designed with the followingcharacteristics:

Planar waveguide Grating Laser Coupling Grating Grating thickness pitchwavelength angle length spacing (nm) (nm) (nm) (degrees) Cladding indesign (mm) (mm) 50-1000 200-500 400-1000 0-40 or Water/SiO2/Polymer0.05-0.5 0.2-50 greater mm

FIG. 4 shows an example of coupling of a laser beam into and out of aplanar waveguide 400 using gratings 402 and 404. The gratings 402 and404 can have the same coupling angle, or different coupling angles, fora given wavelength of light. For example, the gratings 402 and 404 canhave the same or different grating periods. The gratings 402 and 404 arepositioned so that the space on the planar waveguide 400 matches thesize of the imaging sensor that will be used when imaging samples (e.g.,genetic material). The image shown in FIG. 4 can be captured using adifferent camera or other image sensor than what is intended to beutilized when the planar waveguide 400 and the gratings 402 and 404 areused in operation. For example, the image can be captured with a 2Dcharge-coupled device camera. The planar waveguide 400 can include anysuitable material, including, but not limited to, those described inother examples herein. The gratings 402 and 404 can include any suitablematerial (the same or different from each other), including, but notlimited to, those described in other examples herein.

An area 406 on the grating 402 is the reflection generated by a laserbeam being directed at the grating 402. The laser beam impinges on thegrating 402 at a coupling angle so that the laser beam is at leastpartially coupled by the grating 402 and enters the planar waveguide 400as a line 408. In some implementations, the laser beam can have anapproximately elliptical cross section. For example, the laser beam canhave a greatest dimension that is on the order of about 100 μm, butsmaller or larger sizes can be used. In some implementations, a smallerbeam size can provide a greater tolerance of the coupling angle. Forexample, on some gratings a 100 μm laser beam can have a coupling angletolerance of about 0.5 degrees. An area 410 on the grating 404corresponds to the laser beam of the line 408 exiting the grating 404.

For example, the line 408 can be used to illuminate a sample in aflowcell to which the waveguide 400 and the gratings 402 and 404 areapplied. The line 408 can be on the order of about 8-150 μm wide in someimplementations. The narrowest area of the line 408 (sometimes referredto as a waist) can be on the order of about 7 μm. This can be consideredin relation to the line width specifications of some existing sequencingsystems. For example, one existing system may specify a line width of3-8 μm, another system may specify a line width of 10-24 μm, and yetanother system may specify a line width on the order of 40 μm. In someimplementations, beam propagation loss can be less than 10% over adistance of about 1 mm, which can be an approximate width of the planarwaveguide 400 in the x-direction. For example, the planar waveguide canbe about 1.2 mm wide and up to about 100 mm long. This can defineuniformity of about 90% in-line illumination for some detectors (e.g., aTDI scanner). An angle tolerance in the y-direction can be greater thanabout ±3 degrees on a flowcell.

In use, the image sensor that captures imaging light from the flowcellcan be arranged and configured so that its field of view includes theline 408 while not including the areas 406 and 410, in order to avoid orreduce the occurrence of laser light in the image. In someimplementations, the field of view can be a narrow sliver that coversessentially only the line 408 within the planar waveguide 400. Forexample, the field of view can have a width of about 10-24 μm. Forexample, the field of view can have a length of about 800 μm, 1000 μm or2000 μm.

Beams of different wavelengths can be spatially differentiated on theflowcell. In some implementations, a green laser beam, say, shouldimpinge at a different location on the grating 402 than, say, a redlaser beam. For example, this can eliminate or reduce crosstalk betweensignals from the respective laser beams. The green laser beam can thenbe spaced apart from the red laser beam in the y-direction. For example,an area 406′ on the grating 402 schematically illustrates incidence ofthe green laser beam. For example, a line 408′ schematically shows thecoupling of the green laser beam into the planar waveguide 400,essentially parallel with the line 408. For example, an area 410′ on thegrating 404 schematically illustrates coupling of the light of the line408′ out of the planar waveguide 400

The grating 402 and/or 404, and other gratings described herein, can bedesigned to have one or more symmetric coupling angle. FIG. 5 shows anexample graph 500 of coupling angle versus grating period. Couplingangle is marked against the vertical axis and grating period (in nm) ismarked against the horizontal axis. Four beams are illustrated, two ofthem transverse electric (TE) mode beams and the other two transversemagnetic (TM) mode beams. In this non-limiting working example, one TEbeam and one TM beam had a 532 nm wavelength. The other TE beam and theother TM beam had a 660 nm wavelength. The waveguide included Ta₂O₅ of a110 nm thickness, and the substrate included a glass material with arefractive index of about 1.5 and with low autofluorescence. A coverwater buffer had a refractive index of about 1.34 for imaging.

The graph 500 shows simulation results for coupling angle versus gratingperiod. Multiple angles of symmetric coupling can be identified. Forexample, three grating periods allow symmetric coupling, 342 nm, 354 nmand 358 nm. The symmetric coupling angles at those grating periods are+−13.72, +−16.85 and +−8.79 degrees, respectively, indicating that thediffraction can be in either direction from a normal of the grating. Assuch, the graph 600 illustrates that a system with more than one lightsource, and at least first and second wavelengths, can be used with agrating with symmetric coupling angle(s) for the first and secondwavelengths. Additional parameters can impact the coupling efficiencyand the coupling angle. For example, this can include waveguidethickness, grating depth, shape, the refractive index of the waveguide,cladding and/or substrate, the wavelength, and beam polarization.

Known physical relationships, including, but not limited to, a generalwave equation, can be used for further analyzing the propagation ofelectromagnetic waves in the materials. For example, simulations can beperformed and used as a basis for designing a planar waveguide oranother component.

FIG. 6 shows an example graph 600 of coupling angle tolerance analysis.A normalized coupling efficiency is marked against the vertical axis,and an incident beam relative angle in degrees is marked against thehorizontal axis. An x-tilt angle tolerance of 0.57° can be achieved bysmaller beam size. Higher tolerance can be provided by further reducingbeam diameter but increasing position alignment sensitivity. Thecoupling angle tolerance shown in the graph 600 can be about ten timesthe stage tilting resolution, and can show low risk on scan imaging.

FIG. 7 shows an example graph 700 of simulated angle tolerance versusbeam waist. If an implementation were to be very sensitive to theincident angle of the illuminating light, then the line in the planarwaveguide can become brighter or dimmer as the sample is moved duringthe scanning. In the graph 700, a coupling efficiency is marked againstthe vertical axis, and a coupling angle in the substrate is markedagainst the horizontal axis. Four example beams are marked in the graph700, having decreasing beam waists 30 μm, 15 μm, 8 μm and 8 μm (with 30nm grating depth between peak and trough), respectively. The graph 700shows that coupling angle tolerance increases with decreasing beamwaist. The graph 700 also shows that the coupling efficiency of asmall-size beam can still reach over 35% after optimization of gratingdepth. To improve angle tolerance, small beam modeling and highnumerical aperture objective lens for imaging can provide a small beamdiameter without significant cost or challenges.

FIG. 8 shows an example graph 800 of intensity across a waveguide.Intensity is marked against the vertical axis and the position acrossthe planar waveguide (e.g., in form of a column number) is markedagainst the horizontal axis. The graph 800 shows that there can beuniformity at more than 90% of the waveguide. For example, this can be a600 μm region at a center of the waveguide. Edge background in the graph800 can be due to dye solution artificial effects.

Samples can be tested to evaluate planar waveguide illumination for linescan imaging. Such samples can include, but are not limited to,fluorescent beads, a dye molecule carpet, and clusters of geneticmaterial. FIG. 9 shows an example image 900 constructed from line scans.The image 900 can be constructed from multiple line scans. The image 900has a size of 800 μm×800 μm. The scanning can be performed over 1.6 mmat multiple locations and/or lanes. The image 900 is shown forillustrative purposes, and in an actual situation the height can be onthe order of about 75 mm or 100 mm, to name just two examples.

The image 900 shows uniform beads and background. The illumination wasuniform along a long scan region from about 100 μm up to about 5 mmwithout adjustment of the coupling angle or position. At one edge of theimage 900 grating background is presently visible. A design can beprovided with a margin to address this situation. For example, a gutterof about 100 μm can be used for a grating coupler in a flowcell with aplanar waveguide without significant cost or other disadvantage.

FIG. 10 shows an example image 1000 of fluorescence dye molecules. Theimage 1000 is taken of high density fluorescence dye molecules on top ofa polymer layer. Irregularities in the image 1000 are not necessarilydue to the illumination or imaging technique. Rather, the image 1000 canvisualize interface details. For example, the image 1000 can showwaveguide defects, surface patterns and/or other defects in the polymerlayer or a glass substrate, and/or bubbles or dust on a grating.

FIG. 11 shows an example image 1100 of a laser beam shape within awaveguide. A photobleaching pattern in the image 1100 can show a laserbeam shape within a waveguide.

FIGS. 12-15 show example images 1200, 1300, 1400 and 1500 of clusters ofgenetic material. The images have very low background and high SNR atlower power. In some implementations, the power can be on the order of40 times lower than a setup of an existing system. For example, theexisting system may involve 280 mW of laser power and the image 1200 maybe taken at 3.1 mW of laser power. The illumination is uniform acrossthe full field of view of the image sensor and across the scanning.

The image 1200 is 800 μm wide and shows the exit of a planar waveguideplate. The images 1300-1500 are zoomed in compared to the image 1200.The image 1300 is taken at 11 mW with an SNR of 314. The image 1400 istaken at 3.1 mW with an SNR of 154. The image 1500 is taken at 0.4 mWwith an SNR of 35.

The SNR analysis shows that much lower power may be involved for thesame SNR or signal compared to an existing system using the sameanalysis model. One hypothesis can be that background light is muchhigher at the same signal level compared to standard imaging. Anotherhypothesis can be that an equivalent SNR (compared to standard imaging)can be achieved at lower signal level.

The SNR analysis can show that at a signal equivalent to an existingsystem, the planar waveguide can show eight times reduction inbackground level, a two times improvement in SNR and/or a 30 times lowerpower use. This corresponds to the ability to lower the laser power in asystem. The SNR analysis can also show that an SNR equivalent to that ofan existing system can be achievable with a two times lower signal, a 70times reduction in laser power, and/or lower DNA photodamage and laserinduced objective damage or contamination.

In short, up to about a 20-70 times reduction in laser power can beachieved. This can lower some accounting metrics relating to theinstrument, such as the cost of goods sold. This can de-risk laserdamage to optics or the flowcell and objective contamination. The signalto background ratio can be improved up to about eight times. This canprovide improved data quality. For example, a potential for enablingsmall clusters can be enabled. A laser exposure dose can be reduced byabout two to four times. This can provide a reduction in DNAphotodamage.

FIG. 16 schematically shows a system 1600 for laser illumination andfluorescence imaging. The system 1600 can be used with any components,layouts or techniques described herein. The system 1600 includes lasers1602 and 1604. In some implementations, the lasers 1602 and 1604 cangenerate light of different wavelengths. For example, laser light can bepropagated through one or more optical fibers (not shown) in some aspectof the system 1600. The system 1600 includes mirrors 1606 and 1608. Forexample, the laser 1602 can direct light at the mirror 1606, and thelaser 1604 can direct light at the mirror 1608. The system 1600 includesprisms 1610 and 1612. For example, the mirror 1606 can direct light atthe prism 1610, and the mirror 1608 can direct light at the prism 1612.The system 1600 can include a mirror 1614. In some implementations, themirror 1614 has a piezoelectric actuator. For example, the prisms 1610and 1612 can both direct respective light at the mirror 1614. In someimplementations, one or more alignment targets 1616 can be positioned inthe path of a laser beam. For example, the alignment target 1616 caninclude an aperture for laser light. The mirror 1614 can direct thelaser beams at a tube lens 1618. For example, the angle of therespective laser beam as it propagates between the mirror 1614 and thetube lens 1618 can define the coupling angle of that laser beam at adownstream component, such as one or more gratings.

The components 1602-18 can be installed on a suitable bench or othersurface, such as an optics table or equivalent. The components 1602-18can collectively be referred to as excitation optics, or alternativelyas a planar waveguide plate. The following are characteristics orparameters that can apply to a planar waveguide plate: a diode laser(e.g., the laser 1602 and/or 1604) of 532/660 nm wavelength can be used;35/50 mW laser effect can be applied; an optical fiber single mode canbe used. A piezo mirror (e.g., the mirror 1614) can have vertical and/orhorizontal actuation; can optimize coupling angles into a tube lens(e.g., the tube lens 1618) and a flowcell grating; and can be positionedon a back focal plane of a tube lens. Linear translators or mirrors(e.g., the mirrors 1606 and/or 1608) can be used for coarse alignment.

The system 1600 can include a component 1620 that receives light from atleast the tube lens 1618. For example, the component 1620 can becharacterized as an optical structure, or illumination optics, or both.For example, the component 1620 can include a mirror that reflects thelight from the tube lens 1618 in a horizontal direction (e.g., into thedrawing in this view) and another mirror that reflects the light fromthe tube lens 1618 in a vertical direction (e.g., downward in thedrawing in this view). The system 1600 can include an objective lens1622. For example, the objective lens 1622 can include multiple lensesor other optics. The system 1600 can include a sample 1624 that is thetarget of light from the objective lens 1622.

For example, the sample can be provided in a flowcell that has a planarwaveguide and gratings for coupling the light from the objective lens1622. The sample 1624 can be positioned on one or more stages 1626 inthe system 1600. For example, the stage 1626 can include a chuck orother component for mechanically fixating the flowcell during an imagingor scanning operation.

Illumination of the sample 1624 can generate a fluorescent response fromgenetic material in the sample. This response can be characterized asimaging light in the system 1600. The imaging light can enter theobjective lens 1622 can be propagated into the component 1620. Forexample, a dichroic mirror in the component 1620 can direct the imaginglight in another direction, such as toward a mirror 1628. The mirror1628 can direct the imaging light toward a projection lens 1630 in thesystem 1600, after which the imaging light can be incident on acomponent 1632. The component 1632 can redirect and/or condition theimaging light in one or more ways. For example, such conditioning can bepart of a sensing or detection process for the imaging light. Here, thecomponent 1632 includes a mirror that redirects the imaging light towardeither or both of respective image sensors 1634 and 1636. In someimplementations, each of the image sensors 1634 and 1636 can handleimaging light resulting from illumination with light of a particularcolor. For example, the image sensors 1634 and 1636 can be TDI sensors.Other techniques for detection of the imaging light can be used.

The system 1600 can include one or more tracks 1638. In someimplementations, the track(s) can facilitate relative motion between, onthe one hand, at least the sample 1624, and on the other hand, at leastthe objective lens 1622. This can facilitate imaging of a larger area ofthe sample 1624, for example by scanning over the length of one or morechannels in a flowcell. For example, when the image sensor 1634 and/or1636 includes a TDI sensor, the stage 1626 can be a movable stage byvirtue of the track(s) 1638, so that the TDI sensor(s) can scan lineimages of successive areas of the flowcell holding the sample 1624.

Thermal treatment can be applied in the system 1600 or otherimplementations. Because the objective lens 1622 delivers theilluminating light and also captures the imaging light, optical accessto the backside of the sample 1624 may not be necessary. Thermalconditioning can then be applied, at least in part by way of thebackside of the sample 1624. In some implementations, the stage 1626 canbe a thermal stage configured for rapid and/or accurate thermal controlof the sample 1624. For example, the stage 1626 can maintain the sampleat a fixed temperature during an imaging and/or scanning operation.

Dual-surface illumination and imaging can be performed. In someimplementations, gratings can have different locations on top and bottomsurfaces of a flowcell. In some implementations, gratings can bedesigned with different coupling angles based on geometry and/ormaterials. For example, a grating period can be designed differently toprovide proper coupling angles for different situations. For example,top and/or bottom coupling angles can have natural differences due tothe flipping of cladding/substrate. A buffer can be provided between aglass substrate/cover and the illumination from the top at all times.Combinations of two or more of the above approaches can be used.

FIG. 17 shows an example of dual surface imaging using a flowcell 1700.The flowcell can be made from any suitable materials, including, but notlimited to, those mentioned herein. The flowcell 1700 includes asubstrate 1702 (e.g., a top substrate in this view) and a substrate 1704(e.g., a bottom substrate in this view). One or more channels 1706 canbe formed between the substrates 1702 and 1704. For example, thechannel(s) 1706 can contain one or more samples (e.g., genetic material)to be exposed to illumination, such as in a sequencing process. A legend1708 indicates that scanning can be performed in a y-direction and thatan x-direction can then be defined across the flowcell 1700 transverselyto the direction of scanning.

Gratings 1710 and 1712 can be provided on the substrate 1702. In someimplementations, the gratings 1710 and 1712 are provided on a surface ofthe substrate 1702 facing toward the channel 1706 (in this view, on thebottom surface of the substrate 1702). For example, the grating 1710 canbe used to couple a light beam 1714 (which has propagated through thesubstrate 1704 and through the channel 1706) into a planar waveguideformed by the substrate 1702. As such, the light beam 1714 can travelinside the substrate 1702 in essentially the x-direction and illuminatesample material that is on or adjacent the inward (here bottom) surfaceof the substrate 1702. For example, the grating 1712 can be used tocouple the light propagating in the planar waveguide out of thesubstrate 1702.

A similar arrangement can be made with the substrate 1704. Here, agrating 1716 on the substrate 1704 (in this view, on the top surface ofthe substrate 1704) can couple a light beam 1718 that is propagatinginside the substrate 1704 into a planar waveguide formed by thesubstrate 1704, to travel in the x-direction and illuminate samplematerial that is on or adjacent the inward (here top) surface of thesubstrate 1704. A grating 1720 (here also on the top surface of thesubstrate 1704) can be used to couple the light propagating in theplanar waveguide out of the substrate 1704.

Fluorescent responses from the sample materials can then be detected inform of imaging light. An image sensor (not shown) can capture theimaging light triggered at (in this view) the top of the channel 1706 bythe light beam 1714, and can capture the imaging light triggered at (inthis view) the bottom of the channel 1706 by the light beam 1718. Forexample, the light beams 1714 and 1718 can be activated at differenttimes (e.g., alternatingly) so as to allow separate imaging of therespective surfaces. By delivering the light beams 1714 and 1718 throughthe same objective lens that is used for capturing the imaging light,co-registration of the illumination and the imaging at every position inthe y-direction can be obtained,

The light beams 1714 and 1718 can be generated by the same light sourceor by separate light sources. Different wavelengths can be used with theflowcell 1700. In some implementations, both 532 nm and 660 nm laserscan be used for the light beams 1714 and 1718, respectively. Forexample, a 532 nm laser beam can have about a 15 degree coupling angle.With another wavelength, such as 460 nm, a smaller grating pitch and/ora larger coupling angle may be used.

The above example can illustrate approaches that can be taken. In someimplementations, gratings can be at different locations in top andbottom surfaces. The grating 1710 (on the substrate 1702) and thegrating 1716 (on the substrate 1704) are offset from each other in thex-direction. For example, the grating 1710 can be closer to a center ofthe channel 1706. The grating 1712 (on the substrate 1702) and thegrating 1720 (on the substrate 1704) are offset from each other in thex-direction. For example, the grating 1712 can be further from thecenter of the channel 1706. In some implementations, the difference inlocation can be on the order of about 100 μm.

Gratings can have different coupling angles. In some implementations,the grating 1710 (on the substrate 1702) and the grating 1716 (on thesubstrate 1704) can have different coupling angles. For example, a shiftin coupling angle of about 1 degree can be used.

In some implementations, difference in location and difference incoupling angles can both be used. For example, the grating 1710 and thegrating 1716 can have a difference in location in the x-direction ofabout 50 and can have a difference in coupling angle of about 0.5degree.

The above example illustrates that a system can be used with a flowcell(e.g., the flowcell 1700) that includes a first sample surface (e.g.,the surface of the substrate 1702 that faces the channel 1706) parallelto a second sample surface (e.g., the surface of the substrate 1704 thatfaces the channel 1706). A first grating (e.g., the grating 1710) cancouple a first portion of first illuminating light (e.g., the light beam1714) to illuminate the first sample surface. A second grating (e.g.,the grating 1716) can couple a second portion of the first illuminatinglight (e.g., the light beam 1718 when the light beams 1714 and 1718 arefrom the same light source) to illuminate the second sample surface.

The above example illustrates that a first grating (e.g., the grating1710) can be offset, relative to the second grating (e.g., the grating1716), in a travel direction (e.g., the x-direction) of the firstportion of the first illuminating light (e.g., the light beam 1714).

The above example illustrates that a system can be used with a flowcell(e.g., the flowcell 1700) and an image sensor, and that an illuminationarea on the flowcell can be co-registered with a field of view of theimage sensor.

Light that exits the flowcell 1700 can be blocked from entering theobjective lens or from otherwise reaching the image sensor. In someimplementations, a wall 1722 or other barrier can be used. The wall 1722can be referred to as a beam dump. For example, the wall 1722 can bepositioned so as to block the light from the light beam 1714 that thegrating 1712 couples out of the planar waveguide of the substrate 1702.For example, the wall 1722 can be positioned so as to block the lightfrom the light beam 1718 that the grating 1720 couples out of the planarwaveguide of the substrate 1704. In some implementations, the wall 1722can be placed at a location after the objective lens in the path of thereturning light.

FIGS. 18-19 show other examples of dual surface imaging using aflowcell. In FIG. 18, a flowcell 1800 is shown in cross section. Theflowcell 1800 includes a cover glass substrate 1802, a planar waveguide1804, a sample 1806 on or adjacent the planar waveguide 1804, a waterbuffer 1808, a sample 1810 on or adjacent a planar waveguide 1812, and aglass substrate 1814. For example, the cover glass substrate 1802 can bea relatively thin piece of transparent material that allows observationof the sample 1806, such as by way of a microscope. In someimplementations, the planar waveguide 1804 can be considered a topsurface, and the planar waveguide 1812 can be considered a bottomsurface.

A grating 1816 is formed in the planar waveguide 1804, and a grating1818 is formed in the planar waveguide 1812. The gratings 1816 and 1818can have the same or different grating design as each other.

Illuminating light can be coupled into the planar waveguides 1804 or1812. Here, light beams 1820 propagate through the cover glass substrate1802, the planar waveguide 1804 and the water buffer 1808. The lightbeams 1820 are then coupled into the planar waveguide 1812 by thegrating 1818. For example, the light beams 1820 can include one or morelaser beams having a 455 nm, 532 nm and/or 660 nm wavelength. Similarly,light beams 1822 propagate through the cover glass substrate 1802 andare coupled into the planar waveguide 1804 by the grating 1816. Forexample, the light beams 1822 can include one or more laser beams havinga 455 nm, 532 nm and/or 660 nm wavelength. One or more lasers can beused to generate the light beam 1820. One or more lasers can be used togenerate the light beam 1822.

One or more processes can be performed using the flowcell 1800. In someimplementations, a first process involves the top surface (here thesurface of the planar waveguide 1804 facing the water buffer 1808) beingexcited and imaged with the light beams 1822. A second process caninvolve the bottom surface (here the surface of the planar waveguide1812 facing the water buffer 1808) being excited and imaged with thelight beams 1820. Between such processes, refocusing of an objectivelens, and adjustment of coupling angle, can be performed. The sample1806 (e.g., the top surface) or the sample 1810 (e.g., the bottomsurface) can be imaged before the other.

Other gratings (not shown) can be used to couple light out of one ormore of the planar waveguides 1804 and 1812. In some implementations,another grating can be placed in the planar waveguide 1804 and/or in theplanar waveguide 1812. Such a grating can couple light out of therespective planar waveguide 1804 or 1812. For example, the grating 1816and such an additional grating can form a pair in parallel on the samesurface of the planar waveguide 1804 to define an illumination area fora sensor (e.g., a TDI sensor). For example, the grating 1818 and such anadditional grating can form a pair in parallel on the same surface ofthe planar waveguide 1812 to define an illumination area for a sensor(e.g., a TDI sensor).

Instead of coupling light out of the planar waveguide 1804 or 1812 usingan additional grating, another way of dealing with residual light can beused. In some implementations, the light can be blocked directly on theflowcell. For example, a metal strip and/or an absorption strip can beapplied to the planar waveguide to block the light after the light hasilluminated the samples.

The above example illustrates that a flowcell (e.g., the flowcell 1800)can include a substrate (e.g., the glass substrate 1814) to hold asample (e.g., the sample 1806 and/or 1810). The flowcell can include afirst planar waveguide (e.g., the planar waveguide 1804) to lead firstlight (e.g., light beams 1822) for the sample. The flowcell can includea first grating (e.g., the grating 1816) to couple the first light. Theflowcell can include a second planar waveguide (e.g., the planarwaveguide 1812) to lead second light (e.g., the light beams 1820) forthe sample. The flowcell can include a second grating (e.g., the grating1818) to couple the second light.

The above example illustrates that a system can be used with a firstplanar waveguide (e.g., the planar waveguide 1804) into which a firstgrating (e.g., the grating 1816) couples a first portion (e.g., thelight beams 1822) of first illuminating light, and a second planarwaveguide (e.g., the planar waveguide 1812) into which a second grating(e.g., the grating 1818) couples a second portion (e.g., the light beams1820) of the first illuminating light (when the light beams 1820 and1822 are generated by the same light source).

The sample 1806 and/or 1810 can generate one or more fluorescenceresponses based on the illuminating light. For example, the sample 1806and/or 1810 can include one or more fluorophores that reacts to theilluminating light and generates a response.

FIG. 19 shows a cross section of part of a flowcell 1924. In someimplementations, the flowcell 1924 can include a cover glass substrate1926, a planar waveguide 1928, a water buffer 1930, a planar waveguide1932, and a glass substrate 1934. A grating 1936 is formed in the planarwaveguide 1928, and a grating 1938 is formed in the planar waveguide1932. The gratings 1936 and 1938 can have different coupling angles. Thecoupling angle can depend on the grating period, grating depth, and/orthe material of the planar waveguide 1904 or 1912. In someimplementations, the gratings 1936 and 1938 have different gratingperiods. For example, the grating 1936 can have a shorter grating periodthan the grating 1938.

FIG. 20 shows an example image 2000 of flowcell illumination. In someimplementations, the image 2000 can represent an illumination profile ona flowcell, the profile determined by a design model. For example, laserpower can be distributed in a linear area, including, but not limitedto, in a 1200 μm×20 μm area. For example, a laser power of 4 mW can beused.

FIGS. 21A-21B show example flowcharts of a process 2100. The process2100 can be performed in one or more systems, including, but not limitedto, in a sequencing apparatus. For example, the process 2100 can beperformed in any system described herein.

At 2110, at least one sample can be provided. For example, this caninclude obtaining a sample of genetic material, purifying the sample,modifying the sample, and replicating the sample into clusters. Thesample can be provided in a flowcell, including, but not limited to, inany of the flowcells described herein.

At 2120, the flowcell containing the sample can be positioned in asequencing system. For example, in the system 1600 (FIG. 16), the sample1624 can be positioned relative to the objective lens 1622 using thestage 1626 and the track 1638.

At 2130, an objective lens can be focused. For example, the objectivelens 1622 in FIG. 16 can be focused so that the image sensor 1634 and/or1636 obtains a clear view of imaging light from the sample 1624.

At 2140, the sample can be illuminated through the objective lens. Theilluminating light can be coupled into a planar waveguide by a gratingoutside of the field of view of the objective lens. This exampleillustrates that the process 2100 can involve feeding illuminating lightthrough an objective lens and into a flowcell via a grating positionedoutside an image-sensor field of view.

At 2150, imaging light can be captured through the objective lens usingthe image sensor(s).

At 2160, it can be determined if the imaging session is complete. Forexample, if a scanning process is being performed and the entire samplehas not yet been imaged, then the process 2100 can continue to 2170,where a repositioning is performed. The relative position of the sampleand the objective lens is altered. For example, the sample 1624 in FIG.16 can be advanced relative to the objective lens 1622 by the track1638. The process can then return to 2130 for focusing (if necessary)and thereafter illuminate another area of the sample at 2140, and so on.

If the imagining is complete at 2160, the process 2100 can continue to2180, where an image can be constructed. For example, an image can beconstructed from respective line scans taken by a TDI sensor.

FIG. 21B relates to imaging using multiple surfaces in the flowcell. Insome implementations, operations shown in FIG. 21B can be performedinstead of 2130-2150 in the process 2100. At 2130′, an objective lenscan be focused on a first sample surface. For example, this can be thesurface of the planar waveguide 2004 in FIG. 18. At 2140′, the firstsample surface can be illuminated. For example, this can be done usingthe light beams 2021 in FIG. 18. At 2150′, an image of the first samplesurface can be captured using the objective lens.

At 2130″, an objective lens can be focused on a second sample surface.For example, this can be the surface of the planar waveguide 1812 inFIG. 18. At 2140″, the second sample surface can be illuminated. Forexample, this can be done using the light beams 2020 in FIG. 18. At2150″, an image of the second sample surface can be captured using theobjective lens.

The present example illustrates that a method can involve a flowcellthat includes a first sample surface parallel to a second samplesurface, and such a method can further include directing a firstcomponent of the illuminating light (e.g., the light beams 1822) to afirst grating (e.g., the grating 1816) aligned with the first samplesurface, and directing a second component (e.g., the light beams 1820)of the illuminating light to a second grating (e.g., the grating 1818)aligned with the second sample surface.

The present example illustrates that a method can involve adjusting theobjective lens (at 2130′) to focus on the first sample surface inconnection with directing (at 2140′) the first component of theilluminating light to the first grating, and adjusting the objectivelens (at 2130″) to focus on the second sample surface in connection withdirecting (at 2140″) the second component of the illuminating light tothe second grating.

FIG. 22 schematically shows an example of a system 2200 for laserillumination and fluorescence imaging. The system 2200 is configured toperform auto-alignment for planar waveguide illumination. Some aspectsof the system 2200 can be identical or similar to those of the system1600 (FIG. 16). For example, components 1602-1638 of the system 2200 canbe essentially the same components, and/or perform essentially the samefunctions, as the corresponding components in the system 1600.

As mentioned above with reference to FIG. 16, the objective lens 1622will direct illuminating light (sometimes referred to as exciting light)to the sample 1624 and receive imaging light from the sample 1624propagating in essentially the opposite direction toward the component1620. After passing through the planar waveguide, the exciting light canalso enter the objective lens 1622 and propagate toward the component1620. For example, this situation can occur when the planar waveguidehas an exit grating that couples the exciting light out of the flowcell,and no beam dump, etc., is present that could block the exciting lightfrom re-entering the objective lens 1622.

At the component 1620, the imaging light and the returning excitinglight can be separated from each other. For example, a dichroic mirrorcan be used. The imaging light can be directed toward the mirror 1628,ultimately to be received by the image detector(s) 1624-36. Thereturning exciting light, on the other hand, can be directed toward abeamsplitter 2202. The beamsplitter 2202, which is sometimes referred toas a pickoff mirror, redirects a portion of the returning exciting lightarriving from the component 1620 toward a projection lens 2204positioned before an image sensor 2206. In some implementations, thebeamsplitter 2202 has an asymmetrically reflective coating. For example,the respective sides of the beamsplitter 2204 can be coated asdielectric filters.

The image sensor 2206 (e.g., a charge-coupled device) can be configuredfor capturing an image of the sample 1624 including the gratingsthereon, for use in evaluating a quality of the image to be captured bythe image detectors 1634-36. For example, if the sample 1624 is notproperly oriented relative to the camera plane of the image detectors1634-36, then the resulting image can have inferior quality. This can bedescribed as there being a residual between a sample plane and thecamera plane. The image sensor 2206 can then be used in determiningwhether the orientation of the laser beams and/or the sample 1624 shouldbe adjusted, including, but not limited to, by way of the processdescribed below. Such an adjustment or correction can be referred to asde-tilting the sample.

The projection lens 2204 can be used to control the image captured bythe image sensor 2206. The beamsplitter 2202, moreover, has here beenplaced between the tube lens 1618 (sometimes referred to as a projectionlens) and the component 1620. Such a placement can provide the advantagethat the returning exciting light arriving from the component 1620 doesnot pass through the tube lens 1618 before entering the image sensor2206. The magnification, etc. of the returning exciting light istherefore not dependent on the magnification chosen for the tube lens1618.

FIG. 23 shows an example flowchart of a process 2300 for alignment. Forexample, the process 2300 can be performed in a system (e.g., the system2200) to perform auto-alignment of illuminating light relative to aflowcell for improved image quality. More or fewer operations than showncan be performed. Two or more operations can be performed in a differentorder.

At 2310, a flowcell can be positioned. For example, in the system 2200,the sample 1624 can be positioned using the stage 1626.

At 2320, illumination can be provided. For example, in the system 2200one or more laser beams from the lasers 1602 and/or 1604 can be directedthrough the objective lens 1622 and at the sample 1624.

At 2330, excitation light can be captured. The image sensor 2206 in thesystem 2200 can receive light from the beamsplitter 2202 that passesthrough the projection lens 2204. In some implementations, the imagecaptured by the image sensor 2206 can be equivalent to the image of theplanar waveguide 400 in FIG. 4, which image has a sufficiently largefield of view that the gratings 402 and 404 are visible. As describedwith reference to FIG. 4, the area 406 corresponds to the reflection ofthe laser beam impinging on the grating 402. The area 410, moreover,corresponds to the laser beam of the line 408 exiting the grating 404.

The amount of light visible in the areas 406 and/or 410 can be anindication of alignment quality. If the area 406 is relatively brightthis can be an indication that a significant portion of the impinginglaser beam is being reflected (i.e., not coupled) by the grating 402.For example, this can be an indication that the impinging laser beamdoes not have the correct coupling angle and that an adjustment shouldbe done. If the area 410 is relatively dim, or dark, this indicates thatlight is not being coupled into the planar waveguide 400 at the grating402. Similarly, this can be an indication that the impinging laser beamdoes not have the correct coupling angle and that an adjustment shouldbe done.

At 2340, an adjustment of the relative orientation of the flowcell andthe illuminating light can be performed. In the system 2200, theorientation of the laser beam(s) can be controlled by adjusting one ormore of the lasers 1602 or 1604, the mirrors 1606 or 1608, the prisms1610 or 1612, the mirror 1516, the component 1620, and/or the objectivelens 1622. In the system 2200, the orientation of the sample 1624 can beadjusted using the stage 1626. In some implementations, the stage 1626can be adjusted in multiple degrees of freedom. For example, three tiltmotors can be used for exact (e.g., with nanometer precision or greater)positioning of the sample 1624. The adjustment of relative orientationat 2340 can affect the quality of coupling of the impinging laser beaminto the planar waveguide.

At 2350, excitation light can be captured. In some implementations, thiscan be done to evaluate whether the coupling of light into the planarwaveguide 400 (FIG. 4) has improved compared to before the adjustment at2340 was made. In some implementations, if the area 406 has becomedimmer (e.g., having less intensity) in the excitation light captured at2350 than in the excitation light captured at 2330, this can be anindication of improved alignment. In some implementations, if the area410 has become brighter (e.g., having more intensity) in the excitationlight captured at 2350 than in the excitation light captured at 2330,this can be an indication of improved alignment. For example, if thearea 406 has become dimmer and the area 410 has become brighter, thiscan be a particularly significant indication of improved alignment. Assuch, one or more alignment criteria can be applied to the capturedexcitation light.

The capturing of the excitation light can occur at separate moments, asindicated by the operations 2330 and 2350, or it can be donecontinuously, for example in form of a live feed of the image of theflowcell.

At 2360, it can be determined whether the at least one alignmentcriterion has been met. If so, the process 2300 can continue to 2380.For example, the self-alignment can end at 2380.

If the alignment criterion has not been met at 2360, the process 2300can continue with one or more iterations. For example, one or moreadjustments can be performed at 2370 in analogy with the adjustment doneat 2340. The process 2300 can then continue to 2350, where excitationcan be captured for renewed analysis, and so on. The process 2300 cancontinue iterating until the alignment criterion is met at 2360 oranother termination occurs.

FIG. 24 shows an example of a flowcell 2400 with multiple swaths 2402,2404 and 2406. The swaths 2402-06 correspond to the areas where samples(e.g., clusters of genetic material) are present, which samples shouldbe illuminated in an imaging process. The flowcell 2400 includesgratings 2408, 2410, 2412 and 2414. Here, the gratings 2408 and 2410serve as boundaries for the swath 2402; the gratings 2410 and 2412 serveas boundaries for the swath 2404; and the gratings 2412 and 2414 serveas boundaries for the swath 2406. For example, each of the swaths2402-06 can have one or more respective planar waveguides that allowlight to enter at one of the adjacent gratings, travel through theplanar waveguide, and exit at the opposite grating. As such, the swaths2402-06 and the gratings 2408-14 can be placed on the flowcell 2400 toprovide a large imaging area that is compatible with the field of viewof the image sensor (e.g., a TDI sensor). Such a design can saveflowcell area and thereby reduce a consumable cost.

A grating can serve as both an input grating and an exit grating. Forexample, a light beam 2416 is here incident on the flowcell 2400 at thegrating 2410. The light beam 2416 here has the proper coupling angle forthe grating 2410 and is therefore coupled into the planar waveguide ofthe swath 2404. The grating 2412 on the other side of the swath 2404couples the light beam 2416 out of the flowcell 2400. As such, for thelight beam 2416, the grating 2410 can be considered the entry gratingand the grating 2412 can be considered the exit grating.

As another example, a light beam 2418 is here incident on the flowcell2400 at the grating 2412. The light beam 2418 here has the propercoupling angle for the grating 2412 and is therefore coupled into theplanar waveguide of the swath 2404. The grating 2410 on the other sideof the swath 2404 couples the light beam 2418 out of the flowcell 2400.As such, for the light beam 2418, the grating 2412 can be considered theentry grating and the grating 2410 can be considered the exit grating.An imaging process can involve illuminating respective portions of theswaths 2402-06 using corresponding ones of the gratings 2408-16, andcapturing the imaging light accordingly, and then moving the flowcell2400 to instead illuminate another portions of the swaths 2402-06 usingcorresponding ones of the gratings 2408-16, and capturing that imaginglight accordingly.

The terms “substantially” and “about” used throughout this Specificationare used to describe and account for small fluctuations, such as due tovariations in processing. For example, they can refer to less than orequal to ±5%, such as less than or equal to ±2%, such as less than orequal to ±1%, such as less than or equal to ±0.5%, such as less than orequal to ±0.2%, such as less than or equal to ±0.1%, such as less thanor equal to ±0.05%. Also, when used herein, an indefinite article suchas “a” or “an” means “at least one.”

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein. In particular, allcombinations of claimed subject matter appearing at the end of thisdisclosure are contemplated as being part of the inventive subjectmatter disclosed herein.

A number of implementations have been described. Nevertheless, it willbe understood that various modifications may be made without departingfrom the spirit and scope of the specification.

In addition, the logic flows depicted in the figures do not require theparticular order shown, or sequential order, to achieve desirableresults. In addition, other processes may be provided, or processes maybe eliminated, from the described flows, and other components may beadded to, or removed from, the described systems. Accordingly, otherimplementations are within the scope of the following claims.

While certain features of the described implementations have beenillustrated as described herein, many modifications, substitutions,changes and equivalents will now occur to those skilled in the art. Itis, therefore, to be understood that appended claims are intended tocover all such modifications and changes as fall within the scope of theimplementations. It should be understood that they have been presentedby way of example only, not limitation, and various changes in form anddetails may be made. Any portion of the apparatus and/or methodsdescribed herein may be combined in any combination, except mutuallyexclusive combinations. The implementations described herein can includevarious combinations and/or sub-combinations of the functions,components and/or features of the different implementations described.

What is claimed is:
 1. A system comprising: an objective lens; a firstlight source to feed first illuminating light through the objective lensand into a flowcell to be installed in the system, the firstilluminating light to be fed using a first grating on the flowcell; anda first image sensor to capture imaging light using the objective lens,wherein the first grating is positioned outside a field of view of thefirst image sensor.
 2. The system of claim 1, wherein the flowcell isinstalled in the system.
 3. The system of claim 2, wherein the firstgrating and a second grating are positioned on the flowcell.
 4. Thesystem of claim 3, wherein the first and second gratings have differentcoupling angles.
 5. The system of claim 4, wherein the first and secondgratings have different grating periods.
 6. The system of claim 3,wherein the flowcell includes a first sample surface parallel to asecond sample surface, the first grating to couple a first portion ofthe first illuminating light to illuminate the first sample surface, andthe second grating to couple a second portion of the first illuminatinglight to illuminate the second sample surface.
 7. The system of claim 6,further comprising a first planar waveguide into which the first gratingcouples the first portion of the first illuminating light, and a secondplanar waveguide into which the second grating couples the secondportion of the first illuminating light.
 8. The system of claim 6,wherein the first grating is offset, relative to the second grating, ina travel direction of the first portion of the first illuminating light.9. The system of claim 3, wherein the first and second gratings arepositioned on opposite sides of a planar waveguide that illuminates theflowcell, the first grating coupling the first illuminating light intothe planar waveguide, and the second grating coupling the firstilluminating light out of the planar waveguide.
 10. The system of claim9, further comprising a wall that blocks the first illuminating lightcoupled out of the planar waveguide by the second grating from enteringthe objective lens.
 11. The system of claim 2, wherein the flowcell hasmultiple swaths bounded by respective gratings including the firstgrating, and wherein the system uses at least one of the respectivegratings both as an entry grating and an exit grating.
 12. The system ofclaim 2, wherein a waveguide material of the flowcell includes Ta₂O₅.13. The system of claim 1, wherein the first illuminating lightcomprises a first light beam of a first wavelength, the system furthercomprising a second light source to feed second illuminating lightthrough the objective lens, the second illuminating light comprising asecond light beam of a second wavelength.
 14. The system of claim 13,the second light source to feed the second illuminating light throughthe objective lens into the flowcell via the first grating, the firstgrating having a symmetric coupling angle for the first and secondwavelengths.
 15. The system of claim 13, the first light sourcedirecting the first light beam at a first side of the objective lens,and the second light source directing the second light beam at a secondside of the objective lens opposite the first side.
 16. The system ofclaim 13, further comprising a first mirror and a tube lens positionedafter the first and second light sources and before the objective lens,wherein respective angles of the first and second light beams inpropagation from the first mirror to the tube lens reflect correspondingincident angles of the first and second light beams on the firstgrating.
 17. The system of claim 16, further comprising a second mirrorpositioned after the first light source and before the first mirror, thesecond mirror to provide a spatial separation of the first and secondlight beams on the first grating.
 18. The system of claim 1, furthercomprising a second image sensor that captures images of at least thefirst grating and a planar waveguide in the flowcell, wherein the systemevaluates the images using an alignment criterion.
 19. A flowcellcomprising: a substrate to hold a sample; a first planar waveguide tolead first light for the sample; a first grating to couple the firstlight; a second planar waveguide to lead second light for the sample;and a second grating to couple the second light.
 20. The flowcell ofclaim 19, wherein the first grating is positioned on the first planarwaveguide, and wherein the second grating is positioned on the secondplanar waveguide.
 21. The flowcell of claim 19, wherein the first andsecond gratings have different coupling angles.
 22. The flowcell ofclaim 21, wherein the first and second gratings have different gratingperiods.
 23. The flowcell of claim 19, wherein the flowcell includes afirst sample surface parallel to a second sample surface, the firstgrating to couple the first light to illuminate the first samplesurface, and the second grating to couple the second light to illuminatethe second sample surface.
 24. The flowcell of claim 23, wherein thefirst grating is offset, relative to the second grating, in a traveldirection of the first light.
 25. The flowcell of claim 19, the firstgrating to couple the first light into the first waveguide, the secondgrating to couple the second light into the second waveguide, theflowcell further comprising a third grating to couple the first lightout of the first planar waveguide, and a fourth grating to couple thesecond light out of the second planar waveguide.